Genetic variety identifying method in hops

ABSTRACT

This invention is a method for identifying hop varieties based on polymorphism in genetic sequences of hop varieties. Specifically, polymorphic regions of hop DNA are amplified by performing a polymerase chain reaction using an identifying primer designed to amplify regions comprising polymorphism in the base sequences of different varieties, and the amplified DNA fragments are analyzed. In this way, hop varieties can simply and accurately identified.

FIELD OF THE INVENTION

This invention relates to a method of identifying hop varieties, and inparticular, to an identifying method which utilizes genetic engineeringtechnology.

BACKGROUND OF THE INVENTION

Hops gives beer its unique aroma and bitterness, and also helps tosediment out excess protein and suppress bacterial proliferation.However, as the extent to which hops act in this way varies mainlyaccording to the variety, it is necessary to identify hop varieties inorder to manufacture beer of consistent quality and develop new beers.

Conventionally, the identification of hop varieties was based on theirconstituent components, e.g. bitter components (α acid/β acidcohumulone/humulone, colupulone/lupulone) and essential oil components(farnesene, caryophyllene).

However, in addition to the fact that this identifying method based onbitterness and oils requires a great amount of effort for measuring allthe components, it was also unable to determine the varieties preciselybecause, even for the same variety, these components vary widelydepending on the harvesting location and year. Due to recent progress ingenetic engineering, various attempts have been made to elucidatesequences of chromosomes depending on the plant species, and based onthe base sequences found, to identify strains from differences ingenetic information between different plants or varieties of the sameplant, i.e. from the polymorphism of DNA sequences.

An identification of plant variety based on this kind of polymorphism isreliable because the DNA sequence itself is not easily affected byenvironmental influences, and is therefore invariant.

In this regard, the present invention provides a reliable and simplemethod of identifying hop varieties using the aforesaid genetictechnique.

DISCLOSURE OF THE INVENTION

The method of identifying hop varieties according to this inventiondetects this polymorphism genetically based on differences in DNAsequences between varieties. Specifically, portions of the DNA sequenceof hops which show polymorphism are amplified by a polymerase chainreaction (referred to hereafter as PCR) using an identifying primer, andthe varieties are then identified by analyzing this amplified DNA.

To use the above method, it is necessary to understand the polymorphismof DNA between hop varieties.

The term polymorphism specifically covers differences in the geneticsequence due to insertions, deletions or substitutions. The length ofthe sequence involved may be 1 bp, or several tens of bp or more, but ispreferably in a range from several bp to several tens of bp.

This sequential polymorphism between varieties may be detected bydetermining sequences of parts corresponding to chromosome DNA for eachvariety, and then performing a comparative analysis on the sequences. Asimpler method however is to use the RAPD (Random Amplified PolymorphicDNA) technique developed by Williams et al (Nucleic Acids Research, Vol.18, p. 6531, 1990).

This RAPD technique detects polymorphism between unknown DNA sequencesusing PCR. Specifically, primers of low specificity comprising arelatively short synthetic oligonucleotide of about 10 bases are mixedwith a plurality of DNA types, and PCR is performed.

When the DNA sequence of the hop comprises a sequence which is fully orpartially complementary to the primers, the primers anneal with the fullor partial complementary sequence, and the portion enclosed betweenprimers is synthesized and is amplified. When there are differences inthe sequence such as insertions or deletions in the DNA within theportion enclosed between primers, PCR amplified fragments of differentsize are obtained depending on the variety. Further, when one varietycomprises a sequence with which a primer anneals and another varietydoes not, PCR amplified fragments are obtained only for the firstvariety. The polymorphism may then be detected by fractionation, e.g. byelectrophoresis.

The RAPD method may be used not only to detect polymorphism according tothis invention as described above, but also to select primers that candetect polymorphism by PCR, i.e. as a primer designing method.

All polymorphic regions in the DNA of hop varieties detected by the RAPDmethod are addressed by the method of this invention. Also, all primerswhich can amplify polymorphic regions detected by the RAPD method may beutilized as identifying primers according to this invention.

The identifying primer of this invention developed by the above methodmay be one type of synthetic oligonucleotide, or two or more types ofsynthetic oligonucleotide.

The length of this synthetic oligonucleotide is within the range 6-40and preferably 10-21. Specifically, it is convenient to use anoligonucleotide comprising the base sequence indicated by SEQ.ID No.:1-14 in the sequence listing.

A second method for designing an identifying primer according to thisinvention comprises the following steps.

This method first determines the base sequence of the amplified fragmentobtained by the polymerase chain method using an identifying primer(e.g. a primer which can amplify the polymorphic region detected by thePAPD method above, referred to hereafter as a primary primer). Finally,two identifying primers are synthesized which respectively complementpositions a predetermined interval apart in the above polymorphicsequence fragment, so as to produce an amplified fragment which permitsthe polymorphic sequence to be identified by a primary primer with highreproducibility and simplicity.

In other words, according to the above method, the identifying primermay be selectively designed based on the polymorphic sequence andsurrounding sequences.

The aforesaid primary primer may be an oligonucleotide comprising a basesequence described in SEQ.ID Nos: 1-14 of the sequence listing.

For example, the first identifying primer sequence may be designed sothat either one or both primers comprise all or part of the polymorphicsequence mentioned above.

When strain identification is performed by the aforesaid primers, and astrain comprising the selected polymorphic sequence is used, DNAamplification takes place due to PCR as the strain comprises thecomplementary sequence, and a specific amplified DNA is obtained. On theother hand, when a strain not comprising the polymorphic sequence isused, amplified DNA is not produced even when PCR is performed as thestrain does not comprise the complementary sequence. In this casetherefore, it is possible to identify the strain from the presence orabsence of the amplified fragment.

The sequence of the second identifying primer may be designed such thatthe two types of primer are situated respectively upstream anddownstream of the polymorphic sequence.

When strain identification is performed using the above identifyingprimers, amplified DNA having a different internal base sequencedepending on the strain, and in some cases a different size, is producedby PCR. The strain may then be identified from the migration patternobtained by fractionating the amplified DNA, using electrophoresis,electrophoresis after digesting with a restriction enzyme if necessary,denaturing gradient gel electrophoresis or temperature gradient gelelectrophoresis.

A third identifying primer may be designed to comprise a primary primersequence at the 5' end, the base sequence in a polymorphic area joinedto this sequence comprising 5-20 bases linked together. Strainidentification using this identifying primer is useful when for examplethere is a polymorphic sequence at a position to which the primaryprimer is complementary. In other words, the polymorphic sequence may bedetected with greater specificity than by the primary primer alone byattaching the sequence of 5-20 bases to it.

Further, any identifying primer not comprising this type of sequence mayalso conveniently be used as a hop identifying primer provided that itis designed according to the above method.

Specifically, the identifying primer designed according to the abovemethod may be a synthetic oligonucleotide comprising a base sequencedescribed in SEQ.ID Nos. 15-40 of the sequence listing, or a suitablecombination of two synthetic oligonucleotide each comprising part ofthis base sequence, as may be appropriate.

Alternatively, the identifying primer may be a synthetic oligonucleotidewhich comprises part of the base sequence of genome DNA between aposition where the base sequence of SEQ.ID No: 15 (17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39) is annealed to genome DNA, and a positionwhere the base sequence of SEQ.ID No: 16 (18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40) is annealed to genome DNA.

According to this method of designing the second identifying primer(i.e. with this composition), it is possible to determine the basesequence of a region which exhibits polymorphism, and hence the methodprovides a primer which can identify strains more reliably.

In this way, by performing a comparative analysis of DNA between strainsbased on genetic techniques according to the strain identifying methodof this invention, hop strains may be reliably identified without anyeffect from the environment where they were harvested.

This invention will now be described in more detail with reference tospecific examples, but it should be understood that the invention is inno way limited to these examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing a migration pattern obtained when part ofthe DNA of Hallertauer Tradition (HT) and Shinshu Wase (SW) wererespectively amplified using a primary primer (one type of primer: B72),and the amplified fragments were subjected to electrophoresis accordingto the second identifying primer design method.

FIG. 2 is a diagram showing a base sequence of amplified fragmentssubjected to electrophoresis in the vicinity of 600 bp indicated by thearrow in FIG. 1; the upper group shows the base sequence of HT (SEQ IDNO: 43) and the lower group shows the base sequence of SW (SEQ ID NO:44). Identical bases at positions corresponding to HT in the upper groupare shown by a black dot (∘), and where there is a base substitution atpositions corresponding to both, the base is shown. Positions wherethere are no bases corresponding to either type, i.e. missing positions,are shown by a minus sign (-).

The frame enclosure labeled B72WF2 contains a base sequence described inSEQ.ID No: 27 of the sequence listing which is selected as anidentifying primer. The frame enclosure B72WR2 contains a base sequencedescribed in SEQ.ID No: 28 of the sequence listing which is acomplementary sequence of an identifying primer.

FIG. 3 is a photograph showing the results of electrophoresis tofractionate amplified fragments when PCR is performed using anidentifying primer comprising B72WF2 (SEQ.ID No: 27) and thecomplementary sequence of B72WR2 (SEQ.ID No: 28) shown in FIG. 2.

FIG. 4 is a drawing showing a base sequence of another polymorphicamplified fragment (RAPD marker) (SEQ ID NO:45).

FIG. 5 is a drawing showing a base sequence of yet another polymorphicamplified fragment (RAPD marker) (SEQ ID NO:46).

DETAILED DESCRIPTION OF EMBODIMENT

In the hop strain identifying method according to this invention,polymorphic regions, i.e. portions of DNA where the DNA sequence isdifferent for different hop varieties, are amplified by PCR using anidentifying primer, and the strains are identified by analyzing thedifference between amplified fragments.

COLLECTION OF HOP DNA

Hop DNA for study may be collected from minute amounts of hop leaves,cones and pellets.

Hop DNA may be collected by any method generally used to recover DNAfrom plant specimens, for example the typical DNA extraction methodgiven in Nucleic Acids Res., 8, 4321 (1980), or the extraction may beperformed more simply using a commercial BLOOD AND CELL CULTURE DNA KIT(QIAGEN Inc.). When the kit is used, the DNA is absorbed in a negativeion exchange resin column and recovered, so problems due to admixture ofsubstances that might interfere with the reactions may be resolved.

Some methods of designing an identifying primer that may be used in thisinvention will now be described.

FIRST IDENTIFYING PRIMER DESIGN METHOD

To design the primer, any method may be used which searches foroligonucleotide that detect polymorphic regions or sequences indifferent varieties by PCR, but the RAPD method is particularlyconvenient.

To perform the RAPD method, a primer group comprising a plurality ofprimers is first prepared.

The primers used in the ordinary RAPD method may be obtained with a DNAautomatic synthesis kit using, for example, the phosphoamididetechnique. They have random sequences, and their length is 6-40 mer ormore preferably 10-21 mer.

The base sequence disclosed by Williams et al (Nucleic Acids Research,Vol. 18, p.6531, 1990), Beck's common primer or a commercialoligonucleotide from Operon 10-mer Kits made by the OPERON Co. may alsobe employed. PCR is then performed on the different types of hop DNAusing one or more primers from the group prepared above, under the sameconditions as those of the "PCR reaction" described hereafter.

Subsequently, the fragments amplified by PCR are fractionated on asuitable electrophoresis gel, and the migration degree of the amplifiedfragments is compared between strains.

As a result of the above comparison, amplified fragments which are foundto be different or found to have a size difference between strains aretaken as polymorphism amplifying fragments (RAPD markers), and one ortwo primers producing these fragments are selected from the above primergroup to be used as identifying primers.

Examples of such an identifying primer selected in this way is asynthetic oligonucleotide comprising a base sequence described in SEQ.IDNos: 1-14 of the sequence listing, and these sequences may of coursealso be used as complementary sequences.

Any oligonucleotide comprising part of the base sequence shown by SEQ.IDNos: 1-14 of the sequence listing may be used in the PCR reaction as anidentifying primer provided it can amplify any desired polymorphicsequence of hop DNA.

Further, depending on the PCR reaction conditions, any nucleotidecomprising a base sequence similar to that of the aforesaid syntheticoligonucleotide may also be used.

This first design method makes it easy to design an identifying primerthat can detect polymorphism in hops containing unknown sequences.

Moreover, the identifying primer designed by this method fulfills itsfunction very well as will be shown in the following examples.

SECOND IDENTIFYING PRIMER DESIGN METHOD

The second identifying primer design method determines their basesequence of polymorphic amplified fragments (RAPD marker) detected inthe first identifying primer design method and detects polymorphicsequences.

Sequences which can produce amplified fragments containing a polymorphicsequence are selected as the identifying primer from the sequences inthe polymorphic amplified fragments determined here.

A primer of the type listed in "PCR technology", ed. Henry A. Erlich,Takara Shuzo Co., Ltd. which is often quoted in PCR, may be designed.

Specifically, when selecting a primer sequence from the DNAamplification fragment shows size variations as a RAPD marker, anoligonucleotide comprising sequences situated on either side of aninsertion or deletion in the RAPD marker base sequence is selected.

An example of such an identifying primer is a combination ofoligonucleotide comprising the base sequences described in SEQ.ID Nos.27, 28 of the sequence listing.

The size variation of the RAPD marker is from 1 bp to several hundredbp, but a range of 10 bp to several tens of bp is more convenient foridentification. There may also be base substitutions which can beidentified by restriction enzymes or the like even if there is no sizevariation.

The size variation which can easily be distinguished by electrophoresisdepends on the type and concentration of gel used, but as a rule it maybe 1/10 or more of the total length. For example, the primer is designedso that when the insertion sequence is 20 bp, the full length of theamplified product is 200 bp or less. Therefore, by using PCR with thistype of identifying primer, amplified fragments are obtained which makesize variations due to polymorphism easy to distinguish.

Alternatively, when the amplified fragments show size variations betweenvarieties, oligonucleotide may be chosen comprising internal sequencesat insertion sites and sequences at positions bridging sites where thereare deletions. Therefore, by using PCR with this type of identifyingprimer, amplified fragments are obtained for varieties having insertionsand/or deletions.

When the RAPD marker can identify the presence or absence of specificDNA amplification bands depending on the strain, an oligonucleotidecomprising an optimum sequence for the polymerase chain reaction isselected from the internal base sequence of the RAPD marker as primer.An example of such a primer are oligonucleotide comprising the basesequences described in SEQ.ID Nos: 35, 36 of the sequence listing.

Further, when it appears that there is polymorphism only at the positionwherein the primary primer anneals, a synthetic oligonucleotidecomprising a primary primer sequence at the 5' terminus and comprising5-20 of the RAPD marker base sequences, may be selected.

For example when a primer comprising internal base sequences of the RAPDmarker is used and PCR amplified products of the same size are producedfor all varieties, the specificity can be enhanced by designing theprimer in this way. As a specific example, a primer comprising the basesequences described in SEQ.ID Nos: 15 and 16 of the sequence listing mayalso comprise the base sequence described in SEQ.ID No: 12 of thesequence listing at the 5' terminus with base sequences joined to it(FIG. 5). However, when the base sequence in the RAPD marker to bejoined is too short, non-specific amplification bands increase in PCRwhich make detection of polymorphism impossible. On the other hand whenit is too long, primers anneal to every strain regardless of thepresence or absence of polymorphism so that PCR products are producedand detection of polymorphism is again impossible.

When there are recognition sites for restriction enzymes in the RAPDmarker base sequence which would enable identification of varieties sothe primer can be designed to obtain PCR amplified products which retainthese enzyme recognition sites. Example of such a primer areoligonucleotide comprising the base sequences described in the SEQ.IDNos: 33, 34 of the sequence listing.

When the primer is designed in this way, the annealing temperature maybe set when PCR is performed so that only the target fragment (RAPDmarker) are amplified. The identification of DNA amplification bands istherefore easy, and highly reliable results are obtained.

Synthetic oligonucleotide provide a simple means of obtaining theprimers designed in the way described above. The syntheticoligonucleotide used in this invention may be obtained using acommercial DNA autosynthesis machine with, for example, thephosphoamidide method.

The chain length of this oligonucleotide is 15-40, but more preferably20-30. An oligonucleotide comprising any of the base sequences describedin SEQ.ID Nos: 1-40 of the sequence listing may conveniently be used.

In addition to the above, a synthetic oligonucleotide may also be usedwhich comprises part of the hop DNA base sequence between positions towhich other synthetic oligonucleotide comprising two kinds of basesequences among the base sequences described in SEQ.ID Nos: 15-40 of thesequence listing (e.g. 15 and 16, 17 and 18 . . . 35 and 36, etc.), arecomplementary.

The identifying primers according to the aforesaid second design methodamplify the polymorphic region to permit a suitable identification basedon polymorphic amplified fragments, i.e. sequences surrounding thepolymorphic. sequences, and also comprise sequences which are suitablefor PCR. A precise strain identification can therefore be made usingthese primers.

PCR REACTION

Next, the polymorphic region in hop DNA is amplified by PCR usingidentifying primers designed according to the first or second designmethod above.

The PCR reaction is, for example, disclosed by Saiki et. al, Science,vol. 230, p.1350-1354. Specifically, the reaction comprises thefollowing steps: a step for denaturing template DNA, a step forannealing a primer with template DNA, and a step for performing a DNAreplication cycle comprising an extension step by DNA polymerase withthe primer as the starting point.

The PCR reaction was performed by treating each strain in a separatereaction tube. The reaction solution was prepared by adding one or twotypes of synthetic oligonucleotide, DNA polymerase, 4 kinds ofdeoxyribonucleotides (dATP, dTTP, dCTP, dGTP), DNA of each hop varietyas a template DNA, and an amplifying buffer solution (comprising approx.1.0 μM to approx. 4.0 μM but preferably approx. 1.5 μM to approx. 3.0 μMof magnesium chloride, potassium chloride, gelatin, bovine serumalbumin, a surfactant (e.g. Tween 20, NP-40, Triton X-100 (allcommercial names), and dimethylsulfoxide). The reaction tube containingthis reaction solution was set in a thermocycler or the like, and theaforesaid DNA replication cycle was performed a suitable number ofcicles, e.g. approx. 20 cicles to approx. 50 cicles but preferablyapprox. 25 cicles to approx. 40 cicles.

The PCR reaction steps may be performed, for example, under thefollowing conditions.

The denaturing step is normally performed by heating from 90° C. to 95°C. but preferably from 94° C. to 95° C., for approx. 1 min to approx. 3min but preferably for approx. 1 min to approx. 2 min.

The primer annealing step is normally performed by incubating with theprimer from 30° C. to 50° C. but preferably from approx. 35° C. toapprox. 42° C., for approx. 1 min to approx. 3 min but preferably forapprox. 1 min to approx. 2 min. The identifying primer may be one type,or a combination of two or more types, as desired.

The DNA polymerase extension step is performed in the presence ofthermostable DNA polymerase, normally from approx. 70° C. to approx. 73°C. but preferably from approx. 72° C. to approx. 73° C., and fromapprox. 1 min to approx. 4 min but preferably from approx. 2 min toapprox. 3 min. This thermostable DNA polymerase may be commercialthermostable DNA polymerase manufactured by PERKIN ELMER Ltd.

The desired amplified DNA may be obtained by repeating the above steps.

ANALYSIS OF AMPLIFIED FRAGMENTS

The amplified DNA produced by the PCR reaction using the aforesaididentifying primers is fractionated by electrophoresis which is theusual method of fractionating DNA. The strains may then be identifiedbased on the migration pattern obtained.

In electrophoresis, a suitable migration pattern may be obtained byusing approx. 3% to approx. 20% polyacrylamide gel for DNA fragments of1000 deoxyribonucleotides pairs or less, and approx. 0.2% to approx. 2%agarose gel for longer DNA fragments.

The buffer solution used for electrophoresis may be a Tris-phosphoricacid system (pH 7.5-8.0), Tris-acetic acid system (pH 7.5-8.0) or aTris-boric acid system (pH 7.5-8.3), but is preferably a Tris-phosphoricacid system. EDTA may also be added if necessary.

The electrophoresis conditions are different depending on the size ofthe electrophoresis apparatus, but may, for example, be 50-300V for10-120 min, and preferably 150 V for 30 min. As a size markersimultaneously subjected to electrophoresis as a comparison, acommercial marker such as 100 Base-Pair Ladder (Pharmacia Inc.) may beused.

The amplified DNA may be visually detected by a substance such as aphenanthridine dye, for example ethidium bromide, which also interactswith nucleic acids. The staining technique is either to first add asubstance such as ethidium bromide so as to give a final concentrationof approx. 0.5 mg/ml, or to immerse the gel after electrophoresis in anaqueous solution of ethidium bromide containing approx. 0.5 mg/ml forapprox. 10 to 60 min. When the stained gel is irradiated by UV light of254 nm or 366 nm in a dark room, the migration pattern may be detectedas red bands where DNA is combined with ethidium bromide. It will beappreciated that when the staining solution is added to theelectrophoresis apparatus, this migration pattern may be visuallyobserved even during electrophoresis.

In addition to this electrophoresis method, the amplified DNA may beanalyze by any means which can detect its presence or absence, or size.

STRAIN IDENTIFICATION

Strains are identified by a comparative analysis of migration patternsobtained as described above. The comparative analysis may be performed,for example, based on differences in the presence or absence, ordifferences in the size, of predetermined amplified DNA among varieties.

The presence or absence of amplified DNA in a specific variety showswhether the primer used for PCR comprises an annealed sequence (i.e.complementary sequence), and size differences of amplified DNA show thatthere are polymorphic sequences such as deletions or insertions in theregion amplified by PCR depending on the variety.

To perform a more precise identification of strains, it is preferablenot to rely on the result of only one PCR, but to compare migrationpatterns of amplified fragments when one or two differentoligonucleotide are used as identifying primers.

Moreover, the precision with which varieties can be identified and thecapacity to distinguish between them may be improved by observing theresults obtained when the conditions of the PCR are varied, e.g.annealing temperature, magnesium concentration in the reaction buffersolution, etc.

The hop strain identifying method according to this invention, byperforming the above sequence of operations, can clearly distinguishdifferent varieties.

APPLICATIONS

The variety identifying method of this invention can be applied toverify the purity of hop varieties used for hop products such as hoppellets.

For example, a study is made of the difference between the amplified DNAobtained from standard hops and the amplified DNA obtained from hoppellets. If amplified DNA is detected apart from that obtained from thestandard hops, it may be determined that other varieties or species aremixed with the standard hops in the pellets.

When other varieties or species are expected to be present, the amountof amplified DNA, the extent to which these other varieties or speciesare present, i.e. the purity, may be measured by observing, for example,the intensity of the coloration due to ethidium bromide described above.

In this case also, the precision of the purity assessment may beenhanced by using two or more of the synthetic oligonucleotide accordingto this invention in conjunction.

It may be expected that the oligonucleotide of this invention may beapplied to the identification of species in plants other than hops (e.g.mulberries, strawberries, cherries, etc.). Positions where thedeoxyribonucleotides sequence is different in close species arepositions where DNA mutations occur easily, and they are thereforelimited. For example, it is reported that the position for coding rDNAmay be used to identify species of bacteria (E. coli, lactic acidbacteria) or plants (rice seedlings, oranges). Therefore, positionswhere a difference was found between close species according to thisinvention may probably also be used to identify strains in anotherplant.

The variety identifying method according to this invention performs ananalysis based on polymorphism of sequences among varieties, and cantherefore make an accurate distinction between hop varieties unaffectedby environmental factors. Further, the variety identifying methodaccording to this invention may be used not only for identifyingvarieties, but also for measuring the purity of different hop varietiesin hop products.

EMBODIMENT

1-12 show a hop strain identification using an identifying primerdesigned by the first identifying primer design method.

EXAMPLE 1

Extraction of genome DNA

The hops used were Brewer's Gold (referred to hereafter as strain No.1), Northern Brewer (referred to hereafter as strain No. 2), Tettnanger(referred to hereafter as strain No. 3), Saazer (referred to hereafteras strain No. 4), Hersbrucker spaet (referred to hereafter as strain No.5), Spalter select (referred to hereafter as strain No. 6), Hallertauertradition (referred to hereafter as strain No. 7), Shinshu Wase(referred to hereafter as strain No. 8) and Furano Ace (referred tohereafter as strain No. 9).

Green leaf tissue (raw weight Ig) from the above varieties was finelychopped, and the tissue fragments frozen by immersing in liquidnitrogen. After converting the frozen substance to a powder in liquidnitrogen using a Polytron, genome DNA was extracted from 50 mg of thepowder using a BLOOD AND CELL CULTURE DNA KIT (QIAGEN Inc.). 10-20 mg ofgenome DNA was finally obtained for each variety.

EXAMPLE 2

Classification of hop varieties was performed by PCR witholigonucleotide described in SEQ. ID Nos: 1 and 2 as primer. Apolymerase chain reaction was performed in microtube containing 50 μMKCl, 1.5 μM MgCl₂ and 10 μM Tris-HCl buffer solution (pH 8.8) containing0.1% Triton X-100. To the tube were added one unit of thermostable DNApolymerase (Wako Pure Chemicals), 20 nanomoles of four bases (dATP,dTTP, dCTP, dGTP) and 0.1 mg of each hop variety genome DNA prepared inExample 1, 33 picomoles of the oligonucleotide described in SEQ.ID Nos:1 and 2 as primer. The final amount of reaction solution was 30 ml andapprox. 20 ml of mineral oil was added to the tube to preventevaporation of the reaction solution.

The above polymerase chain reaction was performed under the followingconditions. First, after maintaining the temperature at 94° C. for 3min, a denaturing step was performed by heating at 94° C. for 1 min, anda primer annealing step was performed by incubating at 35° C. for 1 min.A DNA polymerase extension step was performed by carrying out 35treatment cycles with thermostable DNA polymerase at 72° C. for 2 mineach, and maintaining the temperature at 72° C. for 10 min. The productwas stored at 4° C.

The amplified DNA obtained by the above polymerase chain reaction wasseparated by electrophoresis at 150 V for 30 min in 100 μM Tris-boricacid buffer solution (pH. 8.0) containing 2 μM EDTA using 5%polyacrylamide gel. 100 Base-Pair Ladder (Pharmacia Inc.) was used as asize marker.

After electrophoresis, the gel was immersed in a 0.5 mg/ml aqueoussolution of ethidium bromide for 10 min, and irradiated in a dark roomwith UV at 254 nm. A red band was detected corresponding to a compoundof DNA with ethidium bromide. The results obtained are shown in Table 1.

As is clear from the table, when synthetic oligonucleotide comprisingthe deoxyribonucleotides sequences described in SEQ.ID Nos: 1 and 2 wereused as primer, two amplified genome bands were detected at approx. 520bp and approx. 530 bp. From the presence or absence of these bands, ninehop varieties were classified into two categories.

    ______________________________________                                               SEQ. ID Nos.                                                                  1, 2    3, 4      5, 6   7, 8 9                                               Amplified DNA (bp)                                                     Hop strain no.                                                                         520    530    750  850  270  370  950  1200                          ______________________________________                                        1               ◯                                                                             ◯                                                                      ◯                                                                           ◯                      2               ◯                                                                             ◯                                     3               ◯                                                                        ◯            ◯                 4               ◯                                                                        ◯            ◯                 5               ◯                                                                        ◯                                                                      ◯                                                                           ◯                           6               ◯                                                                             ◯                                     7               ◯                                                 8        ◯      ◯                                                                      ◯                                                                           ◯                      9               ◯                                                                        ◯                                                                      ◯                                                                      ◯                                                                           ◯                      ______________________________________                                    

EXAMPLE 3

The method was identical to that of Example 2 except that syntheticoligonucleotide described in SEQ.ID Nos: 3 and 4 were used as.primer,and the primer annealing step in the polymerase chain reaction wasperformed at 40° C. The results are shown in Table 1.

When synthetic oligonucleotide described in SEQ.ID Nos: 3 and 4 wereused as primer, two amplified genome bands were detected at approx. 750bp and approx. 850 bp. From the presence or absence of these bands, ninehop varieties were classified into four categories.

EXAMPLE 4

The method was identical to that of Example 2 except that syntheticoligonucleotide described in SEQ.ID Nos: 5 and 6 were used as primer,and the primer annealing step in the polymerase chain reaction wasperformed at 40° C. The results are shown in Table 1.

When synthetic oligonucleotide described in SEQ.ID Nos: 5 and 6 wereused as primer, one amplified genome band was detected at approx. 270bp. From the presence or absence of this band, nine hop varieties wereclassified into two categories.

EXAMPLE 5

The method was identical to that of Example 2 except that syntheticoligonucleotide described in SEQ.ID Nos: 7 and 8 were used as primer.

When synthetic oligonucleotide described in SEQ.ID Nos: 7 and 8 wereused as primer, one amplified genome band was detected at approx. 370bp. From the presence or absence of this band, nine hop varieties wereclassified into two categories.

EXAMPLE 6

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID No: 9 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at40° C. The results are shown in Table 1.

When a synthetic oligonucleotide described in SEQ. ID No: 9 was used asprimer, two ampiified genome bands were detected at approx. 950 bp andapprox. 1200 bp. From the presence or absence of these bands, nine hopvarieties were classified into three categories.

EXAMPLE 7

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID No: 10 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at38° C. The results are shown in Table 2.

When a synthetic oligonucleotide described in SEQ.ID No: 10 was used asprimer, three amplified genome bands were detected at approx. 650 bp,approx. 700 bp and approx. 1200 bp. From the presence or absence ofthese bands, nine hop varieties were classified into four categories.

                  TABLE 2                                                         ______________________________________                                               Hop strain no.                                                                10            11      12                                                      Amplified DNA (bp)                                                     SEQ. ID Nos.                                                                           650     700     1200  1400  550   800                                ______________________________________                                        1                ◯ ◯                                                                       ◯                                                                       ◯                      2        ◯   ◯                                        3        ◯   ◯                                        4                        ◯                                        5                        ◯                                        6                        ◯                                        7                        ◯     ◯                      8        ◯                                                                         ◯                                                                         ◯                                                                       ◯                                                                       ◯                            9                ◯ ◯                                                                       ◯                            ______________________________________                                    

EXAMPLE 8

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID No: 11 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at38° C. The results are shown in Table 1.

When a synthetic oligonucleotide described in SEQ.ID No: 11 was used asprimer, one amplified genome band was detected at approx. 1400 bp andapprox. 1200 bp. From the presence or absence of these bands, nine hopvarieties were classified into two categories.

EXAMPLE 9

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID No: 12 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at38° C. The results are shown in Table 2.

When a synthetic oligonucleotide described in SEQ.ID No: 12 was used asprimer, two amplified genome bands were detected at approx. 550 bp, andapprox. 800 bp. From the presence or absence of these bands, nine hopvarieties were classified into four categories.

EXAMPLE 10

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID No: 13 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at38° C. The results are shown in Table 3.

When a synthetic oligonucleotide described in SEQ.ID No: 13 was used asprimer, four amplified genome bands were detected at approx. 500 bp, 640bp, 650 bp and 140 bp. From the presence or absence of these bands, ninehop varieties were classified into five categories.

                  TABLE 3                                                         ______________________________________                                                 SEQ. ID Nos.                                                                   13               14                                                          Amplified DNA (bp)                                                   Hop strain no.                                                                           500      640    650    1400 540                                    ______________________________________                                        1          ◯   ◯                                                                        ◯                                                                      ◯                          2          ◯   ◯                                                                             ◯                          3                          ◯                                                                             ◯                          4                          ◯                                      5                          ◯                                                                        ◯                               6                          ◯                                                                        ◯                               7          ◯   ◯                                                                             ◯                          8                   ◯ ◯                                                                      ◯                          9          ◯   ◯                                                                        ◯                               ______________________________________                                    

EXAMPLE 11

The method was identical to that of Example 2 except that a syntheticoligonucleotide described in SEQ.ID NO: 14 was used as primer, and theprimer annealing step in the polymerase chain reaction was performed at38° C. The results are shown in Table 3.

When a synthetic oligonucleotide described in SEQ.ID No: 14 was used asprimer, one amplified genome band was detected at approx. 540 bp. Fromthe presence or absence of these bands, nine hop varieties wereclassified into two categories.

EXAMPLE 12

Hop pellets sold as strain no. 8 (manufactured by the Northern HopAgricultural Cooperative, Iwate-ken under license from Sapporo BreweriesLtd.) were crushed in a mortar to a powder and from 20 mg of the powderand approx. 5 mg of genome DNA was extracted using BLOOD AND CELLCULTURE DNA KIT (QIAGEN Inc.). The same procedure was applied to thisDNA as that of Example 2 using synthetic oligonucleotide comprising thedeoxyribonucleotides sequences described in SEQ.ID Nos. 1 and 2 asprimer.

The difference between the detected amplified genome DNA and theamplified genome DNA obtained from standard hops of variety no. 8 wasexamined to verify purity.

Next, a hop strain identification using an identifying primer designedaccording to the second identifying primer design method will bedescribed in Examples 13-30.

EXAMPLE 13

Extraction of genome DNA

Green leaf tissue (raw weight 1 g) from the above varieties was finelychopped, and the tissue fragments frozen by immersing in liquidnitrogen. After converting the frozen substance to a powder in liquidnitrogen using a Polytron, genome DNA was extracted from 50 mg of thepowder using a BLOOD AND CELL CULTURE DNA KIT (QIAGEN Inc.). 10-20mg ofgenome DNA was finally obtained for each variety.

EXAMPLE 14

Selection of RAPD marker

For obtaining RAPD marker, PCR was performed with 0.34 μM of primer-B72(FIG. 2) as primer.

A polymerase chain reaction was performed in a microtube containing 50CMKCl, 1.5 μM MgCl₂ and 10 μM Tris-HCI buffer solution (pH 8.8) containing0.1% Triton X-100. To the microbe tube were added 0.25 units of Taq DNApolymerase (Nippon Gene K. K.), 200 μM each of four bases (DATP, dTTP,dCTP, dGTP) and 17.5 ng of each hop variety genome DNA prepared inExample 13. The final amount of reaction solution was 10 ml.

The above polymerase chain reaction was performed under the followingconditions. First, after maintaining the temperature at 94° C. for 1min, a denaturing step was performed by heating at 94° C. for 30seconds, and a primer annealing step was performed by incubating at 33°C. for 1 min. A DNA polymerase extension step was performed by carryingout 35 treatment cycles with thermostable DNA polymerase at 72° C. for30 seconds each, and maintaining the temperature at 72° C. for 1 min.

The amplified DNA obtained by the above polymerase chain reaction wasseparated by electrophoresis at 150 V for 30 min in 100 μM Tris-boricacid buffer solution (pH. 8.0) containing 2 μM EDTA using 5%polyacrylamide gel. Marker 9 (Nippon Gene) was used as a size marker.

After electrophoresis, the gel was immersed in a 0.5 mg/ml aqueoussolution of ethidium bromide for 10 min, and irradiated in a dark roomwith UV at 254 nm. A red band was detected corresponding to a compoundof DNA with ethidium bromide. The results obtained are shown in FIG. 1.

From FIG. 1, it is seen that the size of the bands in the positionsshown by the arrow is different for HT and SW. The position of the bandshown by the arrow corresponds to the RAPD marker.

EXAMPLE 15

The RAPD marker prepared in Example 14 was cut out from theelectrophoresis gel, introduced into a dialysis tube, subjected to avoltage and eluted from the gel.

Recognition sequences of the restriction enzyme BglII or PstI were addedto the RAPD marker obtained according to the method described in "PCRTechnology" (ed. Henry A. Erlich, pub. Takara Shuzo Co., Ltd.). Aftersub-cloning with the pUC plasmid using this restriction enzymerecognition sequence, the deoxyribonucleotides sequence was determinedby the dideoxy method and is shown in FIG. 2. In the figure, the dotsymbol (∘) shows the same deoxyribonucleotides as in the upper row, andthe bar (-) shows a missing base. The underlined part is thedeoxyribonucleotides sequence of the primer B72, and thedeoxyribonucleotides sequence enclosed by the rectangle is adeoxyribonucleotides sequence to which reference is made in Example 16hereafter.

When sequencing was performed for each band, the insertion of a 32 bpdeoxyribonucleotides sequence was observed in SW as shown in FIG. 2.

EXAMPLE 16

Of the RAPD markers obtained in Example 15, a synthetic oligonucleotidedescribed in SEQ.ID 27 of the sequence listing obtained by referring tothe deoxyribonucleotides sequence B72WF2 enclosed by a rectangle shownin FIG. 2, and the deoxyribonucleotides sequence described in SEQ.ID 28obtained from the complementary sequence B72WR2 were designed(production sub-contracted to Sawaddy Technology).

These synthetic oligonucleotides were used in the following PCR reactionas a primer set. RCR was performed in the 10 ml of reaction solutioncontaining 50 μM MgCl₂ and 10 μM Tris-HCI buffer solution (pH 8.8)containing 0.1% Triton X- 100, to 0.25 units of Taq DNA polymerase(Nippon Gene Inc.), 200 mm of the four bases (dATP, dTTP, dCTP, dGTP),17.5 ng of the genome DNA of each hop strain prepared in Example 13 and0.34 μM of the synthetic oligonucleotides.

The steps in the above PCR were performed under the followingconditions. First, after maintaining the temperature at 94° C. for 1min, a denaturing step was performed by heating at 94° C. for 1 min, anda primer annealing step was performed by incubating at 60° C. for 1 min.A DNA polymerase extension step was performed by carrying out 35treatment cycles with thermostable DNA polymerase at 72° C. for 30seconds each.

The amplified DNA obtained by the above polymerase chain reaction wasseparated by electrophoresis at 150 V for 30 min in 100 μM Tris-boricacid buffer solution (pH. 8.0) containing 2 μM EDTA using 5%polyacrylamide gel. Marker 9 (Nippon Gene) was used as a size marker.

After electrophoresis, the gel was immersed in a 0.5 mg/ml aqueoussolution of ethidium bromide for 10 min, and irradiated in a dark roomwith UV at 254 nm. A red band was detected corresponding to a compoundof DNA with ethidium bromide. The results obtained are shown in FIG. 3.

The same procedure was performed for other varieties, and the resultsare shown in FIG. 3. The other strains used were Fuggle (FU), Cascade(CC), Brewer's Gold (BG), Northern Brewer (NB), Tettnanger (TE), Saazer(SA), Hersbrucker spaet (HE), Perle (PE), Spatter select (SS) and FuranoAce (FA).

As can be seen from the figure, amplification of a 329 bp fragment(arrow) comprising the insertion position was observed for BG, NB, TE,SW and FA. A 299 bp fragment not comprising the insertion position wasobserved for all strains. The strain-specific fragments 600 bp, 700 bp,710 bp were also observed. These fragments are easy to identify and wereobserved with high reproducibility.

EXAMPLE 17

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 15 and 16 were used as primer.The results are shown in Table 4.

From the table, it is seen that when the synthetic oligonucleotidesdescribed in SEQ.ID Nos: 15 and 16 were used as primer, one amplifiedgenome band was detected at approx. 500 bp. From the presence or absenceof this band, twelve hop varieties were classified into two categories.

EXAMPLE 18

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 17 and 18 were used as primer,and the primer annealing step in PCR was performed at 62° C. The resultsare shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 17 and 18 wereused as primer, one amplified genome band was detected at approx. 260bp. From the presence or absence of this band, twelve hop varieties wereclassified into two categories.

EXAMPLE 19

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 19 and 20 were used as primer,and the primer annealing step in PCR was performed at 65° C. The resultsare shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 19 and 20 wereused as primer, two amplified genome bands were detected at approx. 500bp and approx. 550 bp. From the presence or absence of these bands,twelve hop varieties were classified into two categories.

EXAMPLE 20

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 21 and 22 were used as primer.The results are shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 21 and 22 wereused as primer, one amplified genome band was detected at approx. 710bp. From the presence or absence of this band, twelve hop varieties wereclassified into two categories.

EXAMPLE 21

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 23 and 24 were used as primer.The results are shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 23 and 24 wereused as primer, one amplified genome band was detected at approx. 330bp. From the presence or absence of this band, twelve hop varieties wereclassified into two categories.

EXAMPLE 22

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 25 and 26 were used as primer,and after performing PCR, 20 PCR cycles were performed under the sameconditions as those of the previous PCR using 1 ml of reaction solutionas a template DNA. The results are shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 25 and 26 wereused as primer, two amplified genome bands were detected at approx. 160bp and approx. 200 bp. From the presence or absence of these bands,twelve hop varieties were classified into two categories.

EXAMPLE 23

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 29 and 30 were used as primer,and the primer annealing step in PCR was performed at 57° C. The resultsare shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 29 and 30 wereused as primer, one amplified genome band was detected at approx. 350bp. From the presence or absence of this band, twelve hop varieties wereclassified into two categories.

EXAMPLE 24

The method was identical to that of Example 22 except that syntheticoligonucleotides comprising the deoxyribonucleotides sequences describedin SEQ.ID Nos: 30 and 31 were used as primer, PCR was performed twice,and the reaction solution was treated with the restriction enzyme NlaIII(Daiichi Pure Chemicals). The results are shown in Table 4.

When synthetic oligonucleotides comprising the deoxyribonucleotidessequences described in SEQ.ID Nos: 31 and 32 were used as primer, twoamplified genome bands were detected at approx. 220 bp and approx. 360bp. From the presence or absence of these bands, twelve hop varietieswere classified into four categories.

EXAMPLE 25

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 33 and 34 were used as primer,the primer annealing step in PCR was performed at 67° C., and thereaction solution was treated by the restriction enzyme Taq I(Boehringer Mannheim). The results are shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 33 and 34 wereused as primer, two amplified genome bands were detected at approx. 220bp and approx. 270 bp. From the presence or absence of these bands,twelve hop varieties were classified into three categories.

EXAMPLE 26

The method was identical to that of Example 16 except that syntheticoligonucleotides described in SEQ.ID Nos: 35 and 36 were used as primer,and the primer annealing step in PCR was performed at 58° C. The resultsare shown in Table 4.

When synthetic oligonucleotides described in SEQ.ID Nos: 35 and 36 wereused as primer, one amplified genome band was detected at approx. 400bp. From the presence or absence of this band, twelve hop varieties wereclassified into two categories.

EXAMPLE 27

PCR and electrophoresis were performed in the same way as in Example 14using 33 picomoles of Beck's common primer (A25; 5'-GGTCAGGCACCA-3'(SEQID NO:47)). As a result, more than ten amplified genome bands wereobserved from approx. 200 to 2000 bp, and the band at approx. 500 bpwhich was present or absent depending on the strain, was used as a RAPDmarker. When the deoxyribonucleotides sequence of this marker wasexamined, the results shown in FIG. 4 were obtained.

Synthetic oligonucleotides described in SEQ.ID Nos: 19 and 20 wereaccording to the sequences denoted by A and B enclosed by a rectangle inFIG. 4. Oligonucleotides described in SEQ.ID Nos: 37 and 38 weredesigned according to the sequences denoted by C and D which areunderlined in FIG. 4. These oligonucleotides described in SEQ. ID Nos:37and 38 comprise parts of the RAPD marker. These oligonucleotidesdescribed in SEQ ID Nos: 19 and 20 contain primary sequences at5'terminus.

The procedure was identical to that of Example 16 except that 35annealing step cycles at 65° C. were performed for using SEQ ID Nos: 19and 20 as primer, and 30 annealing step cycles at 60° C. were performedfor using SEQ ID Nos: 37 and 38 as primer. As a result when SEQ ID Nos:19 and 20 were used as primer, bands at 500 bp and 550 bp were observedas shown in Table 4, and when SEQ ID Nos: 37 and 38 were used as primer,a band at 459 bp was observed. From the presence or absence of thesebands, twelve hop varieties were classified into two categories.

EXAMPLE 28

PCR and electrophoresis were performed in the same way as in Example 14using 33 picomoles of Beck's common primer (C16; 5'-CGCCCTGCAGTA-3'(SEQID NO:48)). As a result, more than ten amplified genome bands wereobserved from approx. 200 to 2000 bp, and the band at approx. 500 bpwhich was present or absent depending on the variety, was used as a RAPDmarker. When the deoxyribonucleotides sequence of this marker wasexamined, the results shown in FIG. 5 were obtained.

Synthetic oligonucleotides described in SEQ.ID Nos: 15 and 16 were basedon the sequences denoted by A and B enclosed by a rectangle in FIG. 4.Oligonucleotides described in SEQ.ID Nos: 39 and 40 are based on thesequences C and D which are underlined in FIG. 5.

SEQ. ID Nos: 39 and 40 were used as primer and anealing step wereperformed at 60° C., 30 cicle. An am amplified genome band at 500 bp wasobserved for all varieties but the varieties could not be identified. Onthe other hand when an identical procedure was followed using 15 and 16,the amplified genome bands shown in Table 4 were obtained, and from thepresence or absence of these bands, twelve hop strains were classifiedinto two categories.

EXAMPLE 29

From a general overview of Table 4 which summarizes the types inExamples 16-28, it is seen that it is possible to distinguish each of 12variety of hops.

Examples where the fragment size is listed were taken as a reference forvariety identification, and examples where treatment by restrictionenzymes was performed are shown in brackets ().

                                      TABLE 4                                     __________________________________________________________________________    Primer set  Strain                                                            SEQ. ID No.                                                                         (bp)  FU                                                                              CC                                                                              BG NB                                                                              TE                                                                              SA                                                                              HE PE                                                                              SS                                                                              HT                                                                              SW FA                                       __________________________________________________________________________    15/16 500   ◯                                                                     ◯   ◯                                                                      ◯                            17/18 260   ◯                                                                   ◯                                                                      ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                               19/20 500     ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                            ◯                                                                   ◯                                                                    ◯                            19/20 550     ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                            ◯                                                                   ◯                                                                    ◯                            21/22 710          ◯  ◯                               23/24 330       ◯     ◯                               25/26 200   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                            25/26 160   ◯                                                                        ◯                                                                   ◯                                                                          ◯                                                                     ◯                               27/28 710                         ◯                               27/28 700                         ◯                               27/28 600       ◯                                                                    ◯                                                                   ◯   ◯                            27/28 329       ◯                                                                    ◯                                                                   ◯                                                                              ◯                                                                    ◯                            27/28 299   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                            29/30 350   ◯                                                                   ◯                                                                      ◯                                                                   ◯                                                                   ◯                                                                      ◯                                                                   ◯                                                                   ◯                                                                      ◯                            31/32 360(NlaIII)                                                                         ◯                                                                   ◯                                                                          ◯                                                                      ◯                                                                   ◯                                                                   ◯                                                                   ◯                               31/32 220(NlaIII)                                                                         ◯                                                                   ◯                                                                      ◯                                                                   ◯                                                                     ◯                                                                          ◯                                                                    ◯                            33/34 270(TaqI)                                                                           ◯                                                                   ◯                                                                   ◯                                                                      ◯                                                                        ◯                                                                          ◯                            33/34 220(TaqI) ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                               35/36 400     ◯                                                   37/43 459     ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                            ◯                                                                   ◯                                                                    ◯                            39/40 500   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                                                                   ◯                                                                   ◯                                                                   ◯                                                                    ◯                            __________________________________________________________________________

EXAMPLE 30

Shinshu Wase hop pellets (manufactured by the Northern Hop AgriculturalCooperative, Iwate-ken under license from Sapporo Breweries Ltd.) werecrushed in a mortar, and approx. 5 mg of genome DNA was extracted from20 mg of the powder using BLOOD AND CELL CULTURE DNA KIT (QIAGEN Inc.).The purity of the DNA obtained was examined by the same procedure asthat of Example 29 using synthetic oligonucleotides comprising thedeoxyribonucleotides sequences described in SEQ.ID Nos. 15-40 as primer.

Few non-specific amplified bands appeared, and the desired marker couldeasily be verified. Highly reproducible results were also obtained inrepeated purity tests.

FIELD OF APPLICATION

According to the genetic identifying method of this invention, a preciseand simple identification of hop varieties which is unaffected byenvironmental or other conditions, may be made. The genetic identifyingmethod of this invention may also be effectively used to examine thepurity of products in which hops are a raw material.

Hence, by identifying hop varieties, the genetic identifying method ofthis invention may be used to maintain the quality of productscontaining hops at a constant level, or to improve that quality.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 48                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 19               TGC                                                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 19               TGT                                                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               TTGG                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               CCGA                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               TCGC                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               CCCA                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               CAGA                                                       - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ                                      #21                TTAG T                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 19               TGC                                                        - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24CCTG TAAG                                                  - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24ACTT CTAT                                                  - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                 22GCG TA                                                    - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ                                      #               25 AGTT AAGTA                                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ                                      #               25 CTAG CTGGC                                                 - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ                                      #               25 CACC ATCTG                                                 - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ                                      #              26  CATG ACCTTA                                                - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24GGAA GGAA                                                  - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                23CCCA GGC                                                   - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                23GTTG ACC                                                   - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                23AATA CAC                                                   - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               GCTG                                                       - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                 22ACA TA                                                    - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                TAAG C                                                     - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:29:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                 22TGA GG                                                    - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:30:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               CTAA                                                       - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:31:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             28   TAAC AACTTCTT                                              - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:32:(xi) SEQUENCE DESCRIPTION: SEQ                                      #             28   TGTT CTAGAACA                                              - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:33:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                23AGCT GCG                                                   - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:34:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24AATC GTGG                                                  - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:35:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24CCTC TCCC                                                  - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:36:(xi) SEQUENCE DESCRIPTION: SEQ                                      #                24CACT GTCT                                                  - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:37:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                ATTT C                                                     - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:38:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                TCAA G                                                     - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:39:(xi) SEQUENCE DESCRIPTION: SEQ                                      # 20               TACA                                                       - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:40:(xi) SEQUENCE DESCRIPTION: SEQ                                      #21                TTTC G                                                     - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:41:(xi) SEQUENCE DESCRIPTION: SEQ                                      #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 13 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:42:(xi) SEQUENCE DESCRIPTION: SEQ                                      #      13                                                                     - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 599 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:43:(xi) SEQUENCE DESCRIPTION: SEQ                                      - GGCGATTCTG CAAGAGACAC AACGCAGACA AGAAATTTGA ATAACATAAT CG - #AGAGGGGT         60                                                                          - TTGCTCTCGA GTCCCTCCTA GACACCTACA TAGCAATTGC TACAATTTCC TA - #GTGTCCGC        120                                                                          - AAATATTGTA GGGTTACTAA TGGATTTATT GTTTACATCT GTTGCATTCT TT - #TATGTAAA        180                                                                          - TGATGATGAT GAGATTCCAT ATGAATGAGA GTCTTTATAA GCTAAAAATT TA - #ATGGCATG        240                                                                          - CATTGTATCC CAAGGCAAAT GGTCATGCAG ATGCAATGGA GTACTGAATA AA - #TTAAATTA        300                                                                          - AACTGGTTTT ACAGACGCTG TTGACAAACA AAATAGGTAA TACCAGAAGC TT - #ACCTTGCT        360                                                                          - GTCAGGAGCA AATTTTAAAC GAACAGCTTT CCAGTCAGAC ACGTCCTCAT CG - #GTGCCACC        420                                                                          - ACTTCCAGTT TCACTGCTTG GTTCCACAGG TTTCTCAAGT ACGTTCTTCC GG - #AACTTCTC        480                                                                          - TAGTCTTGCT AGAACCTGAA GGAAACGTTA ACCAGCAAAG TTGGTAATTG GA - #AACTTAAT        540                                                                          - TAGCAAATAA TGCTAATGTG AAGAGCAATA CATCAAATTG TTTTATATGC AG - #AATCGCC         599                                                                          - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 629 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:44:(xi) SEQUENCE DESCRIPTION: SEQ                                      - GGCGATTCTG CAAGAGACAC AACGCAGACA AGAAATGTGA ATAACATAAT CG - #AGAGGGGT         60                                                                          - TTGCTCTCGA GTCCCTCCTA GACACCTACA TAGCAATTGC TACAATTTCC TA - #GTGTCTGC        120                                                                          - AAATATTGTA GGGTTACTAA AATATATAGG AAATATCTAA ACGTGTAAAA AA - #TGGATTTA        180                                                                          - TTGTTTACAT CTGTTGCATT CTTTTATGTA AATGATGATG ACGAGATTCC AT - #ATGAACGA        240                                                                          - GTCTTTATAA GCTAAAAATT TAATGGCATG CATTGTATCT CAAGGCAAAT GG - #TCATGCAG        300                                                                          - ATGCAATGGA GTATGGAATA AATTAAATTA AACTGGTTTC ACAGACGCTG TT - #GACAAACA        360                                                                          - AAATAGGTAA TACCAGAAGC TTACCTTGCT GTCAGGAGCA AATTTTAAAC GA - #ACAGCTTT        420                                                                          - CCAGTCAGAC ACGTCCTCAT CGGTGCCACC ACTTCCAGTT TCACTGCTTG GT - #TCCACAGG        480                                                                          - TTTCTCAAGT ACGTTCTTCC GGAACTTCTC TAGTCTTGCT AGAACCTGAA GG - #AAACATTA        540                                                                          - ACCAGCAAAG TTGGTAATTG GAAACTTAAT TAGCAAATAA TGCTAATGTG AA - #GAGCAATA        600                                                                          #           629    ATGC AGAATCGCC                                             - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 500 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:45:(xi) SEQUENCE DESCRIPTION: SEQ                                      - GGTCAGGCAC CATGTACTAG CTGGCGCGAC ACTCCCACTG CACACCTATT TC - #CGGTCCGT         60                                                                          - AGTAGATTAC TTTAACATCT CCCCATTCTA GATCACACCG AATGGGATCC AA - #GCCCTCTC        120                                                                          - TGCGCTATAC ATTCTTTACT TTCTGAATGG TTGGGACGAG CCCACTCCAC AT - #GAGGTGCA        180                                                                          - TTACTTGTTC GATCTCAGGA CCAACCCCTC TCACAACAAC TCAGGCTTTT TC - #CACTTCTA        240                                                                          - TATAGGCATA GGGGGATTAC ATACCTCAAC GGTATTTCTC ATAGGTCGAA TG - #CCGGGAGG        300                                                                          - TATCATAAGG GATACTTCCT CACCTTGGAC ATCGAGGCCA ACAAATTTGG GC - #CTTAACTC        360                                                                          - GTCGGGGTCC ATTTGAGCGA CCATTGCCTA CTGAGGAGAT GTCAATCGGC CA - #AGAGTTGG        420                                                                          - CTAACATGAG TTCTAAAGAT AAGGATGTAA AAGGTTGGTC ACACTTGACC TC - #CTTCAGAT        480                                                                          #500               GACC                                                       - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 488 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       #ID NO:46:(xi) SEQUENCE DESCRIPTION: SEQ                                      - CGCCCTGCAG TACCTTCCTG TAAGGGTTTA CAGGGGACTT GATACAACCT CT - #AGGTGCAA         60                                                                          - TCAAGTTAGC CCTCACAATG GGAGAGAAAC CAAGACAAAC CACTATAATG AC - #AAACTTTG        120                                                                          - TCGTGGTAGA TTGTGCCTCA GCCTTAAATG CGGTATTAGA AAGACCTTCC CT - #AAGAGAAT        180                                                                          - TGAAGGGAAT AACCTCAGTA TAACACTTGG CCATAAAATT CCCAACTCTT GG - #AGGAATAG        240                                                                          - CGAGCGTGAA AGGGGAATAG AAGGAAGCAA GGGAATGTTA TAACACGTCC CT - #CCACACAG        300                                                                          - TCATGAAACT GCCATTACCC ATGGTGATGG TGGTGCATGG AGGTGCAAAC TC - #ACATGACT        360                                                                          - TAGACCCTCG AGTTGTTGAG GAGATCAGAA TCAAAATGGA TAACAGAGAG AT - #AAATGAGC        420                                                                          - TATGCCTAAA AAAATCAGAA ATTAGAAGAG CAGTGCGAAA AATGATAGAA GT - #GCTCTACT        480                                                                          #         488                                                                 - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:47:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "SYNTHETIC DNA"RIPTION: /desc                                              #ID NO:48:(xi) SEQUENCE DESCRIPTION: SEQ                                      #       12                                                                    __________________________________________________________________________

What is claimed is:
 1. A random amplified polymorphic DNA (RAPD) methodfor identifying a variety of the hop plant Humulus lupuluscomprising:(a) providing a variety of the hop plant Humulus lupulus anda primer; (b) isolating DNA from the variety; (c) conducting apolymerase chain reaction using the DNA and the primer; (d) determiningwhether or not the polymerase chain reaction resulted in the productionof a DNA molecule; and (e) identifying the variety if the DNA moleculewas produced, or identifying the variety if the DNA molecule was notproduced.
 2. The method of claim 1, wherein a plurality of primers isprovided, and the polymerase chain reaction is conducted using theplurality of primers.
 3. The method of claim 1, wherein the primer is asynthetic oligonucleotide.
 4. The method of claim 1,wherein the primercomprises a first nucleotide sequence selected from the group consistingof SEQ ID NO:1 to SEQ ID NO:40, or the primer comprises a secondnucleotide sequence comprising the complement of the first nucleotidesequence.
 5. The method of claim 1,wherein the primer comprises a firstnucleotide sequence selected from the group consisting of SEQ ID NO:1 toSEQ ID NO:14, or the primer comprises a second nucleotide sequencecomprising the complement of the first nucleotide sequence.
 6. Themethod of claim 1,wherein the primer comprises a first nucleotidesequence selected from the group consisting of SEQ ID NO: 15 to SEQ IDNO:40, or the primer comprises a second nucleotide sequence comprisingthe complement of the first nucleotide sequence.
 7. A random amplifiedpolymorphic DNA (RAPD) method for constructing a primer for use inidentifying a variety of the hop plant Humulus lupulus comprising:(a)providing a variety of the hop plant Humulus lupulus and a first primer;(b) isolating DNA from the variety; (c) conducting a polymerase chainreaction using the DNA and the first primer, thereby producing a DNAmolecule; (d) determining the sequence of the DNA molecule; (e)constructing a second primer comprising a sequence contained in the DNAmolecule, thereby constructing the primer for use in identifying a hopvariety.
 8. The method of claim 7, wherein a plurality of first primersis provided, and the polymerase chain reaction is conducted using theplurality of first primers.
 9. The method of claim 7, wherein the firstprimer is a synthetic oligonucleotide.
 10. The method of claim 6,wherein the second primer is a synthetic oligonucleotide.
 11. The methodof claim 7,wherein the first primer comprises a first nucleotidesequence selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:14, or the first primer comprises a second nucleotide sequencecomprising the complement of the first nucleotide sequence.
 12. Themethod of claim 7,wherein the second primer comprises a third nucleotidesequence selected from the group consisting of SEQ ID NO: 15 to SEQ IDNO:40, or the second primer comprises a fourth nucleotide sequencecomprising the complement of the third nucleotide sequence.
 13. Themethod of claim 7, wherein the second primer comprises a sequencecontained in the first primer.